猪五种繁殖障碍性疫病病原体多重PCR的建立及初步应用

摘要

To establish the multiple PCR for detecting classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine pseudorabies virus(PRV), porcine circovirus type 2(PCV2) and porcine parvovirus(PPV),five pairs of primers were designed to amplify CSFV E2,PRRSV Nsp2,PRV gB,PCV2 ORF2 and PPV VP2 according to the gene sequences in GenBank of these five viruses.Reaction conditions were optimized to establish the multiplex PCR on the basis of the single PCR.The multiplex PCR had good sensitivity and specificity.To evaluate the multiplex PCR,127 clinical samples were detected for detecting the five viruses,among which 38.6%(49/127) samples were detected for two or more viruses,which means the multiplex PCR can be used as rapid diagnosis for single or mixed clinical samples infected with CSFV,PRRSV,PRV,PCV2,PPV.%为了建立能够同时检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)和猪细小病毒(PPV)的多重PCR,并用于猪场感染情况的动态监控以及临床诊断,根据GenBank中已发表的5种病毒的基因序列,针对CSFV的E2、PRRSV的Nsp2、PRV的gB、PCV2的ORF2和PPV的VP2基因,分别设计了特异性引物.在建立的单项PCR基础上,通过优化反应条件,建立了能同时检测5种病毒的多重PCR,并具有较高的灵敏性和良好的特异性.采用建立的多重PCR对127份疑似病猪的扁桃体活体组织进行检测,检出了5种病毒的存在,其中感染2种及以上病毒的样品比例为38.6%(49/127),表明该方法是一种快速、灵敏、高效的病原学检测手段.